| This project was designed to determine the underlying kinetic mechanism of external tetraethylammonium (TEA+) block of large-conductance Ca2+-activated K+ (BK) channels. BK channels are unique among ion channels in their sensitivity to activation by voltage and a ligand, intracellular Ca2+. Their large conductance makes them an ideal model system for kinetic studies. TEA+ is the prototypical open channel blocker of K+ channels. The theory of open channel block states that the blocker interacts exclusively with the open state of the channel. Upon appropriate stimulation the channel undergoes a conformational change to an open state. In the presence of an open channel blocker, the blocker may interact with its site within the ion-conducting pathway to achieve a temporary block of ion flux. This mechanism has been likened to a cork in a bottle. Although external TEA+ block is generally referred to as open channel block, there have been relatively few detailed studies to confirm that classification.;This study has developed a set of testable predictions based on the simple model of open channel block. These predictions were compared to experimental results obtained from steady-state recording of excised outside-out patches containing single BK channels. Analyses focused on describing the effects of TEA+ on the individual open and closed states of BK channels. BK channels exhibit complex gating comprised of at least five closed and three open states. Externally applied TEA+, at a concentration of 100 muM, caused an ∼45% decrease in the open probability of the channel, reductions of ∼20, 65, and 55% in the lifetimes of the individual open states (from short to long), and decreased both mean burst durations and mean total open time within bursts, by ∼20 and ∼45%, respectively. Of these results, only the reduction in open lifetimes is consistent with a mechanism of simple open channel block. Modeling of subsets of the experimental data across concentrations of TEA+ established that these results were consistent with two different mechanisms: state-independent block, and trapping block. |
